Proteoglycans are widely distributed in the body, and consist of a protein core, to which glycosaminoglycan side chains are covalently linked. The major proteoglycan in cartilage is aggrecan. Collagen and aggrecan are the primary components of the articular cartilage which covers the bones of joints, as well as of other cartilages. Other proteoglycans are also found in skin, tendons, cornea, sclera, intervertebral disc, and elsewhere in the body. They vary in the type and number of the glycosaminoglycan side chains, and in the molecular weight of the protein core. Aggrecan comprises chondroitin sulphate and keratan sulphate side chains.
The proteoglycans, including aggrecan, may be affected in a variety of acute and chronic conditions, including connective tissue diseases such as scleroderma and systemic lupus erythematosus and in degenerative joint conditions, or following trauma. Degenerative joint diseases are frequently referred to as arthritis conditions, and include osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and gout, among others.
Arthritis is a crippling musculoskeletal disease that incapacitates millions of people from all walks of life. The two most common forms of arthritis are osteoarthritis (OA) and rheumatoid arthritis (RA). Osteoarthritis alone is the reason for nearly a quarter of all general practitioner consultations. It is present in more than 50% of people over thirty years of age, and statistics released by the Arthritis Foundation of Australia show that one in five Australians is affected. Moreover, the number of people suffering with arthritis is increasing as a consequence of an increasingly elderly population. It is clear that as the incidence of the major killers such as heart disease, cancer and infectious diseases diminishes due to improved public health care, the impact of chronic diseases such as arthritis falls more heavily on the population and on health care systems.
The major feature of both OA and RA is cartilage degradation and loss of aggrecan, the molecule which gives cartilage its unique weight-bearing properties. Most pathological conditions involving proteoglycan destruction are not detected until the disease is relatively advanced. In particular, the initial presenting features of the major arthritides, osteoarthritis and rheumatoid arthritis, are pain, swelling and stiffness. Symptoms do not manifest themselves until the degree of cartilage destruction is already significant.
Very little is known of the precise mechanisms underlying the changes in cartilage that lead to damage, or about what causes the loss of aggrecan and the eventual eburnation of the articular surface. Clearly there is an urgent need for:
(i) a better understanding of the mechanisms involved in aggrecan loss and cartilage destruction, and PA1 (ii) the development of better diagnostic techniques for detecting the early stages of joint disease before the damage has become so extensive that tissue repair is no longer possible. PA1 (i) MMPs-1, -2, -3 and -9 are synthesised by chondrocytes and synovial cells PA1 (ii) elevated levels of stromelysin and collagenase have been found in joint fluids of OA and RA patients PA1 (iii) synthesis and secretion of MMPs is markedly stimulated both in vivo and in vitro by cytokines such as IL-1 and TNF that are known to promote cartilage resorption.
In order to identify the early stages, it is necessary to find a marker of the disease that is regularly observed in cases that are clinically well-defined. There is a particularly urgent need for a reliable method of very early detection of cartilage damage. Such methods are, of course, also applicable to monitoring of disease progression, monitoring of the efficacy of methods of therapy, and screening new therapeutic agents.
Methods of detecting proteoglycan products in biological samples have been proposed for this purpose. Thus, for example, U.S. Pat. No. 4,778,768 by Heinegard and Lindblad and U.S. Pat. No. 5,185,245 by Heimer disclose methods of detecting aggrecan or fragments thereof in synovial fluid, and their use in monitoring changes in articular cartilage and monitoring therapy. U.S. Pat. No. 5,177,020 by Timpl et al discloses the use of immunoassay of heparan sulphate-proteoglycan in body fluids as a method of diagnosis of diabetes mellitus and its complications, such as nephropathy. Australian patent No. 645742 by Caterson and Hardingham discloses the use in diagnostic assays of a monoclonal antibody directed against altered chondroitin sulphate epitopes present in osteoarthritic cartilage. These antibodies detect epitopes present on abnormal chondrotin sulphate/dermatan sulphate chains. However, none of these methods has found wide application, and it appears that they also may be detecting changes which occur relatively late in the development of disease. Early changes must be identified. Therefore, the basic mechanisms involved in initiation of cartilage destruction have been studied intensively by a number of workers. This has required an understanding not only of the detailed structure of the aggrecan molecule, but also identification of the enzymes which can degrade aggrecan, and the sites at which these enzymes cleave the protein core.
Aggrecan is the major proteoglycan in cartilage, and is responsible for its resilience and load-bearing properties. The properties of aggrecan have been extensively studied, and the sequence of the protein moiety is known for several species. The loss of aggrecan is a major feature of the cartilage degeneration associated with arthritis. Normal turnover and pathological loss of aggrecan from cartilage involves proteolytic cleavage of the core protein at the N-terminus, where two globular domains, G1 and G2, are separated by a short interglobular domain (IGD). This is illustrated in FIG. 3 below. The IGD has been identified as a key site of proteolytic attack; however the proteinases responsible for cleavage in the tissue have not been identified, despite intensive study of a variety of candidate enzymes.
The matrix metalloproteinases (MMPs) have long been regarded as the most likely mediators of cartilage destruction. The different kinds of human MMPs are listed in Table 1. The well-documented but circumstantial evidence that implicates the MMPs in aggrecan degradation is as follows:
TABLE 1 ______________________________________ The Matrix Metalloproteinases Interstitial collagenase MMP-1 EC 3.4.24.7 Neutrophil collagenase MMP-8 EC 3.4.24.34 Collagenase-3 MMP-13 Gelatinase A MMP-2 EC 3.4.24.24 Gelatinase B MMP-9 EC 3.4.24.25 Stromelysin-1 MMP-3 EC 3.4.24.17 Stromelysin-2 MMP-10 EC 3.4.24.22 Stromelysin-3 MMP-11 Matrilysin MMP-7 EC 3.4.24.23 Metalloelastase MMP-12 MT-MMP MMP-? ______________________________________
Our research has focused on the aggrecan IGD, with the aim of determining precisely where enzymes cleave the core protein, and which cartilage enzyme/s are responsible for this cleavage. Using a purified G1-G2 substrate prepared from cartilage aggrecan (1) we have identified cleavage sites specific for all the MMPs except MMP-11, MMP-12, and MT-MMP (2-4, 27), as well as cathepsin B (3) plasmin and urokinase-type plasminogen activator (5). Our results show that all the MMPs cleave at a site located between Asn 341 and Phe 342 (Table 2) (based on the human aggrecan sequence (6)) and that this is the preferred and predominant site of cleavage for this class of enzyme. We have also located a minor MMP cleavage site between Asp 441 and Leu 442; however the incidence of cleavage at this site is low, and not all the MMPs show this activity (3, 4).
However, other studies (7-9) have identified a major cleavage site within aggrecan IGD which was different to the MMP cleavage site, and differed from those characterised for some other candidate proteinases, namely cathepsin B (3), leukocyte elastase (12), plasmin and urokinase (5). These studies have shown that, under conditions of normal and interleukin-1 (IL-1) stimulated turnover, bovine cartilage explants released aggrecan fragments with N-terminal sequences corresponding to cleavage between Glu 373 and Ala 374 (7-9). The amino acid sequences flanking the metalloproteinase and the aggrecanase cleavage sites are given in Table 2.
TABLE 2 ______________________________________ Metalloproteinase and aggrecanase cleavage sites in aggrecan IGD Proteinase Cleavage sites ______________________________________ Matrix metalloproteinase ....D I P E N.sub.341 .dwnarw. F F G V G... Aggrecanase ...I T E G E.sub.373 .dwnarw. A R G S V... ______________________________________
In addition, the major aggrecan fragments found in synovial fluids from OA and joint injury patients result from cleavage at the same Glu 373-Ala 374 bond (10,11). The enzyme responsible for this cleavage has been named aggrecanase, but its identity has remained unknown. With one exception (15,16) all attempts to identify proteinases that can cleave at this aggrecanase site in vitro, or to purify the activity from cartilage or chondrocyte extracts, have failed, despite intense interest from research institutions and the pharmaceutical industry.
It is currently generally accepted in the field that two groups of enzymes are involved in aggrecan breakdown in degenerative joint conditions, namely the matrix metalloproteinase group (Table 1) and the as-yet unidentified but widely accepted "aggrecanase". The cleavage sites for the matrix metalloproteinases and for aggrecanase are different (Asn 341-Phe 342 and Glu 373-Ala 374 respectively), and in most cases the cleavage products are detected by electrophoresis and sequencing.
The identification of aggrecanase-derived fragments in human synovial fluids suggests that "aggrecanase" is the enzyme that is responsible in vivo for loss of aggrecan from cartilage. However, other data indicate that MMPs are also directly involved in aggrecan degradation in vivo: GI fragments with C-terminal sequences that correspond to MMP cleavage have been extracted from and immunolocalized in human articular cartilage (13,14 respectively). These findings suggest that cleavage by both MMPs and aggrecanase is involved in aggrecan degradation in vivo, and readily explain the occurrence of C-terminal . . . DIPEN fragments in cartilage matrix and N-terminal ARGSV . . . fragments in joint fluids (Table 2).
We have previously reported that a metalloproteinase, MMP-8, can cleave a G1-G2 substrate in vitro at the glu.sub.373 -ala.sub.374 site, and therefore has aggrecanase activity (15,16). We have also shown that in vitro MMP-8 cleaves primarily at the MMP site, . . . N.sub.341 .dwnarw.FFGVG . . . , and that cleavage at the aggrecanase site occurs as a secondary event (16). Our data do not imply that MMP-8 is aggrecanase in cartilage, and indeed no MP-8, protein or antigen has been found in cartilage; however, our data do confirm that the metalloproteinases are likely to play a prominent role in aggrecan degradation.
There is one body of thought which considers that the principal event in aggrecan breakdown is cleavage by aggrecanase at Glu 373-Ala 374, and another which considers that cleavage at the MMP site, Asn 341-Phe 342, may be the key event. Collectively, the current available literature would suggest that there is in vivo cleavage at both the MMP and the aggrecanase sites, but it is not known which cleavage is the primary, or rate-determining step.
International patent publication No. WO-93/22429 by Shriners' Hospital for Crippled Children describes the identification of aggrecan fragments in synovial fluid from patients with post-traumatic arthritis caused by recent knee injury, and from patients with early or late stage OA. Biochemical analysis identified a single major N-terminal sequence in all samples, commencing ARGSV . . . . At least two populations of aggrecan fragments, both relatively large and both with the N-terminal sequence ARGSVILXVK . . . were identified. An N-terminal sequence FFGVGGEEDIXVQ . . . was obtained after deglycosylation of chondroitin-sulphate-bearing human aggrecan fragments produced following treatment with stromelysin. These stromelysin fragments showed no evidence of an N-terminal sequence beginning at Ala 374. The inventors proposed a method of monitoring onset or a progression of osteoarthritis comprising assaying biological fluid, such as synovial fluid, for proteoglycan breakdown products, specifically resulting from cleavage of aggrecan between Glu 373 and Ala 374, and in particular where the breakdown product had a N-terminal amino acid sequence ARGSV . . . . While it was suggested that antibody detection was suitable, no specific antibodies were disclosed. It was also proposed that antibodies directed to the aggrecanase cleavage region or enzyme inhibitors comprising the amino acids Glu-Ala could be used for treatment of OA.
At the 40th Annual Meeting of the Orthopaedic Research Society (Feb. 21-24, 1994), monoclonal antibodies were described which are able to detect aggrecan fragments generated by aggrecanase and by metalloproteinase (17-19). monoclonal antibody BC-3 recognises amino-neo-epitope fragment ARGSVIL . . . resulting from digestion of aggrecan by aggrecanase, and antibody BC-4 recognises the carboxy-neo-epitope sequence . . . FVDIPEN resulting from digestion of aggrecan with the metalloproteinase stromelysin (17). Monoclonal antibody BC-3 detects aggrecan peptides with N-terminal sequence ARGSV . . . , which is produced in vivo by the action of aggrecanase, and also in vitro by neutrophil collagenase (matrix metalloproteinase-8; MMP-8) (15,16). A polyclonal antibody recognising the peptide FVDIPEN (SEQ ID NO. 1) detected fragments resulting from the action of stromelysin or gelatinase A (18-19, 28); the antibody requires the sequence VDIPEN (SEQ ID NO. 2) for full recognition of the peptide. This antibody was able to detect aggrecan fragments in synovial fluid (19) and in cartilage (14,19).
There have so far been no reports of any antibody against the carboxy-neo-epitope with N-terminal sequence FFGVG . . . resulting from matrix metalloproteinase action, or that such an antibody could in fact detect aggrecan fragments in biological samples.
The amino acid sequences flanking the metalloproteinase site and the aggrecanase site in the IGD are shown in Table 2 above. The newly-created N- and C-termini generated by proteolysis at these sites represent neo-epitopes with antigenic determinants that are different to the antigenic determinants present on sequences in the intact, undegraded protein. Potentially, neo-epitope antibodies are enormously useful for detecting discrete products of aggrecan degradation. Importantly, in OA cartilage specimens, the . . . DIPEN epitope was specifically immunolocalised in regions exhibiting extensive fibrillation and loss of aggrecan (14), providing in vivo evidence for metalloproteinase involvement in joint disease.
To investigate MMP action further we have developed a novel neo-epitope monoclonal antibody. We have produced a mono-specific hybridoma that secretes IgG.sub.1 specific for the N-terminal FFGVG . . . sequence generated by MMPs. This antibody is diagnostically more useful than the anti-DIPEN antibody, because it detects the FFGVG . . . epitope that is released from the tissue into the synovial space, whereas the . . . DIPEN epitope located on the G1 domain is predominantly retained in the tissue.